This is an exploratory project (R21) that will utilize a mosquito Anopheles gambiae as a model organism to discover genes and gene products involved in mosquito immune responses against arbovirus infections. Arboviruses transmitted by mosquito vectors are of great public health concern worldwide. For example, in the U.S. there were 4,180 clinical West Nile cases with 149 associated deaths in 2006. Other mosquito-borne viruses (e.g., eastern equine encephalitis, western equine encephalitis, La Crosse, and St. Louis encephalitis) infect thousands of people annually. Thus, mosquito-borne viral diseases demand more research to provide intervention strategies in their viral transmission to humans. One area of the life cycle for which we lack knowledge is the response of the mosquito vector to an arboviral infection. Through an understanding of this relationship we will gain the ability to develop new strategies to limit the impact of viral infections in the host. An. gambiae is a major vector for human malaria Plasmodium falciparum in Africa, but it is also a natural vector for O'nyong-nyong virus (ONNV) transmission. Because of its medical importance, the whole genome sequence of An. gambiae is fully determined. With this genome sequence available, An. gambiae offers a powerful model system to study gene expression profiles of mosquito vectors in response to viral infections at the genome level. In general, mosquito vectors are resistant to infections. This suggests that mosquitoes have evolved an active defense mechanism to protect themselves against pathogenic consequences of arboviral infections. In fact, our previous studies have shown that An. gambiae seem to actively modulate ONNV replication through induction of a heat shock cognate protein, hsc-70B. In ONNV- infected An. gambiae, hsc-70B expression was found to be highly induced, and RNA interference (RNAi) gene- silencing of hsc-70B increased ONNV titers in the mosquito. To identify a more comprehensive catalog of antiviral immune genes in a tissue-specific manner, we propose to profile the changes of gene expression in the midgut of ONNV-infected An. gambiae. This project will employ three major techniques, 1) laser capture microdissection (LCM) for RNA and protein extraction from virus-infected midgut epithelial cells, 2) 60- mer oligo-microarrays, and 3) iTRAQ", a quantitative proteomics analysis. Utilizing the above technologies, we hypothesize that differential gene expression between ONNV- infected and uninfected An. gambiae can be quantitatively measured and candidate genes critical for immune responses to ONNV infection in An. gambiae will be identified. Our specific aims are 1) identification of candidate antiviral genes in the midgut of ONNV-infected An. gambiae, 2) quantitative proteomic analysis of ONNV-infection in An. gambiae, and 3) functional characterization of candidate anti- ONNV genes using RNA interference gene silencing. Characterization of these mosquito genes is expected to have a profound impact on widening our understanding of immune responses of An. gambiae to ONNV infection. Furthermore, it is possible that findings of our study will provide the foundation for elucidating a general mechanism for mosquito-arbovirus interactions. To this end, comparative genomics of orthologs of anti-ONNV genes of other arboviral mosquito vectors can be subsequently investigated as an extension of this proposed study. Considering there exist overlapping immune responses against bacterial and malaria infections in An. gambiae, our findings may also provide information applicable to Plasmodium infection.Narrative of the Study Arboviruses (arthropod-borne viruses) transmitted by mosquito vectors are a serious public health concern worldwide. For example, dengue endemic regions have rapidly expanded throughout the world, especially in South America. Domestically, human cases of West Nile virus infections seem to be on the rise each year- for example, there were 4,180 clinical West Nile virus cases with 149 associated deaths in 2006. This number is a sharp increase from the previous year with about 3,000 cases (www.cdc.gov/ncidod/dvbid/westnile). Other mosquito-borne viruses (e.g., eastern equine encephalitis, western equine encephalitis, La Crosse, and St. Louis encephalitis) also infect thousands of people annually in the U.S. Thus, mosquito-borne viral diseases demand more research to provide more effective intervention strategies in viral transmission to humans. In general, mosquito vectors are susceptible to viral infection but only to a certain level, beyond which mosquitoes become resistant to infections, restricting viral titers below cytopathic levels. This phenomenon suggests that mosquitoes may have evolved an active defense mechanism to protect themselves against pathogenic consequences of arboviral infections. In fact, our previous studies have shown that An. gambiae seem to actively modulate o'nyong-nyong virus (ONNV) replication through induction of a heat shock cognate protein, hsc-70B. In ONNV-infected An. gambiae, hsc-70B expression was found to be highly induced, and RNA interference (RNAi) gene-silencing of hsc-70B increased ONNV titers in the mosquito. To identify a more comprehensive catalog of antiviral immune genes in a tissue-specific manner, we propose to profile the changes of gene expression in the midgut of ONNV-infected An. gambiae. This project will employ three major techniques, 1) laser capture microdissection (LCM) for RNA and protein extraction from virus-infected midgut epithelial cells, 2) 60-mer oligo-microarrays, and 3) iTRAQ", a quantitative proteomics analysis. Utilizing the above technologies, we hypothesize that differential gene expression between ONNV- infected and uninfected An. gambiae can be quantitatively measured and candidate genes critical for immune responses to ONNV infection in An. gambiae will be identified. Our specific aims are 1) identification of candidate antiviral genes in the midgut of ONNV-infected An. gambiae, 2) quantitative proteomic analysis of ONNV-infection in An. gambiae, and 3) functional characterization of candidate anti- ONNV genes using RNA interference gene silencing. Characterization of these mosquito genes is expected to have a profound impact on widening our understanding of immune responses of An. gambiae to ONNV infection. Furthermore, it is possible that findings of our study will provide the foundation for elucidating a general mechanism for mosquito-arbovirus interactions. To this end, comparative genomics of orthologs of anti-ONNV genes of other arboviral mosquito vectors can be subsequently investigated as an extension of this proposed study. Considering there exist overlapping immune responses against bacterial and malaria infections in An. gambiae, our findings may also provide information applicable to Plasmodium infection.